The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been 17 instrumental for the development of economically sustainable lignocellulose biorefineries. 18 Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of 19 action remains enigmatic and their activity and stability under process conditions are hard to 20 control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen 21 atoms, we show that H 2 O 2 , and not O 2 as previously thought, is the co-substrate of LPMOs. By 22 controlling H 2 O 2 supply, stable reaction kinetics and high enzymatic rates are achieved, the 23 LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of 24 reductants is alleviated. These results offer completely new perspectives regarding the mode of 25 action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, 26 and industrial biorefining. 27 28 29

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes, broadly distributed across the Tree of Life. We recently reported that LPMOs can use H2O2 as an oxidant, revealing a novel reaction pathway. Here, we aimed to elucidate the H2O2-mediated reaction mechanism with experimental and computational approaches. In silico studies suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation, and guides the derived hydroxyl radical towards formation of a copper-oxyl intermediate. The initial H2O2 homolytic cleavage and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are suggested to be the main energy barriers. Under single turnover conditions, stopped-flow fluorimetry demonstrates that LPMO-Cu(II) reduction to Cu(I) is a fast process compared to the re-oxidation reactions. We found that re-oxidation of LPMO-Cu(I) is 2000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. In agreement with the notion of ternary complex formation, when chitin is added, reoxidation by H2O2 is accelerated whereas that by O2 slows. Simulations indicated that Glu60, a highlyconserved residue, gates the access to the confined active site and constrains H2O2 during catalysis, and Glu60 mutations significantly decreased the enzyme performance. By providing molecular and kinetic insights into the peroxygenase activity of chitinolytic LPMOs, this study will aid the development of applications of enzymatic and synthetic copper catalysis and contribute to understanding pathogenesis, notably chitinolytic plant defenses against fungi and insects.